G-protein coupled receptors (“GPCRs”) are integral membrane proteins that relay signals from cell surface receptors to intracellular effectors. To date, over 1000 different GPCRs have been identified (see, e.g., Gether, 2000, Endocrine Reviews 21:90–113; Kolakowski, 1994, Receptor Channels 2:1–7). GPCRs respond to a broad range of extrracellular signals including, for example, hormones, neurotransmitters, chemokines, odorants and light (see, e.g., Buneman & Hosey, 1999, J. Physiol. 517.1:5–23). They play vital roles in fundamental cell processes including growth, differentiation and survival. Currently, over 25% of drugs currently on the market target GPCRs. These drugs include antipsychotics, antihistamines, antihypertensives, anti-migraine drugs, anti-ulcer drugs and analgesics.
They typically span the membrane seven times and comprise an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The transmembrane domain usually comprises seven transmembrane helices. Members of this superfamily couple to heterotrimeric intracellular G proteins comprising α, β and γ subunits. In its inactive state the Gα subunit binds GDP. When the receptor is activated, it physically associates with a G-protein and catalyzes the exchange of GTP for bound GDP in its Gα subunit. Following receptor activation, the βγ subunits, which bind to each other very tightly, as well as the GTP-Gα subunit dissociate from the receptor and from each other. All three subunits, the activated α and the free βγ, interact with and activate specific downstream cellular effectors, depending on the nature of the receptor-ligand interaction. Intrinsic GTPase activity in the Gα subunit converts it to the inactive GDP-bound form, and reassociation of the heterotrimeric complex restores the receptor to its resting state.
Currently, 17 Gα-subunits, 5 Gβ subunits and 12 Gγ subunits have been identified and described (Dhanasekaran et al., Oncogene 1998 17:1383–1394; Hepler and Gilman, Trends Biochem. Sci. 1992 17:383–387; Strathman et al., Proc. Natl. Acad. Sci. 1991 86:7407–7409). Although signaling heterogeneity is derived from the various combinations of α, β and γ subunits, G-proteins are classified into four distinct classes based on the sequence similarity of their Gα subunits (subunits that are >50% similar are typically grouped in the same class): (a) Gαs (coupled to stimulation of adenylyl cyclase activity and cAMP formation); (b) Gαi (coupled to inhibition of adenylyl cyclase activity and suppression of cAMP formation; (c) Gαq (coupled to activation of phospholipase C (PLC) and mobilization of intracellular calcium); and (d) Gα12: (coupled to activation of downstream effectors such as rac, rho; coupled to changes in intracellular cytoskeletal elements; not clearly understood).
According to conventional techniques, the activities of only a subset of the known GPCRs can be readily measured in a high throughput manner. In particular, those GPCRs that couple with the Gαq effector protein typically generate a calcium signal, mediated by various calcium-mobilizing messengers, that can be measured in a readily accessible assay. In particular, membrane-permeant fluorescent dyes, such as Fura-2 AM, and Fluo-4 AM, have a high affinity and specificity for ions such as calcium and undergo a significant shift in emitted wavelength when bound to calcium. With such dyes, detection of changes in intracellular calcium in whole cells, in real-time, is feasible and affordable. Advances in hardware, fluorescent detection tools and sensitive cameras, have further enabled assays for screening compounds in drug discovery programs in a high throughput format (Grynkiewicz et al., J. Biol. Chem. 260:3440–3450; Mason et al., in “Fluorescent and Luminescent probes for Biological Activity”; ed. W. T. Mason; pp 161–195; Academic Press, London). There are many advantages to using such an assay: (i) the response is rapid (milliseconds to seconds); (ii) the response is transient; (iii) the response can be measured in whole, live cells and not just cell membranes and (iv) the response is a measure of receptor activation, rather than just receptor binding. Modulation of such a response is therefore biologically relevant, and can have long-term consequences to the behavior of the cell.
Although the calcium assay is very suitable for high-throughput screens, it suffers at least one major disadvantage: it can only be applied to receptors that are coupled to second messenger pathways that involve changes in intracellular calcium. For the GPCR superfamily, this technique is restricted to the Gαq-coupled receptors. Traditional methods for assaying the remaining GPCRs are labor and time intensive and cannot be readily utilized in a high throughput manner. These include binding assays, cAMP detection, and reporter gene techniques, and typically involve significantly more time and effort than a calcium mobilization assay. Further, binding assays are not whole cell assays and therefore do not necessarily have a biologically relevant read-out. In addition, cAMP assays are not as rapid and transient as calcium assays. Furthermore, reporter gene techniques are significantly more time intensive, not always robust and reproducible and measure responses that are much further downstream of receptor activation than calcium mobilization. These responses can therefore be prone to receptor-independent cellular effects that can be misinterpreted as receptor-dependent effects. Therefore, new compositions and methods are needed to facilitate high throughput measurement of the activities of a broad range of GPCRs.